Potential ADCC/ADCP Effects of a Humanized Anti-human Cripto-1 Antibody

Background/Aim: Cripto-1 (CR-1) is recognized as a potential theranostic biomarker expressed on the surface of tumor cells, including cancer stem cells. We previously reported a humanized anti-human CR-1 antibody (ah²CR1-Ab35), derived from a phage display library, that demonstrated growth inhibition in cancer-derived cells. In this study, the potential of this antibody was evaluated for its ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in CR-1–expressing cells. Materials and Methods: Jurkat cells recombinantly expressing CD16a, low-affinity CD16a, and CD32a, in which cytoplasmic signaling was engineered using a luciferase reporter system, were employed as effector cells to target MDA-MB-231, MIA-Paca-2, LS 174T, and HEK293 cells, in which CR-1 expression was enhanced using CR-1 expression plasmids. Anti-sense CR-1 expression plasmids were also constructed and independently introduced into each cell line to establish background controls by suppressing endogenous CR-1 expression. Photon counts from luciferase reactions stimulated by the anti-CR-1 antibody were then measured after subtracting background-derived signals. Results: Both ADCC and ADCP effects mediated by the anti-CR-1 antibody were successfully demonstrated in a dose-dependent manner. The antibody exhibited an EC₅₀ of approximately 1 μM in MDA-MB-231 cells and 1-4 μM in MIA-Paca-2 cells, whereas in LS 174T cells, the EC₅₀ ranged from 1 to 4 μM for ADCC but was not clearly determined for ADCP owing to the low photon counts. Conclusion: The humanized anti-human CR-1 antibody has been suggested to mediate ADCC and ADCP effects in CR-1–expressing cells. However, these effects did not appear to correlate with CR-1 expression levels. The efficacy of CR-1 targeting via ADCC and ADCP effects may depend on the characteristics of the target cells.